Immunological tolerance: one of biology's • • • • most Intriguing mysteries

THOMAS E. STARZL, MD, PHD, AND NORIKO MURASE, MD

Why is it that so many organ recipients, especially re­cipients of a liver, are able to survive without taking immunosuppressive drugs? The first recipient of a

liver at Baylor, Amie Garrison, was 5 years old at the time of her transplantation in 1984. When she was age 15, however, she rebelled and refused to continue taking her medication. We feared the worst, but happily she has survived off drugs for more than 5 years. Furthermore, she is now married and has a young child of her own. What happened?

Survival without immunosuppressive medication is not un­common. Indeed, at our clinic in Pittsburgh we deliberately wean liver transplant recipients from immunosuppressive therapy un­der close supervision (1, 2 )-quite a different situation than noncompliance. We believe that patients who can survive with stable graft function off immunosuppressive therapy have genu­ine immunological donor-specific tolerance and can anticipate a normal life expectancy.

Another dramatic case also illustrates my point. Approxi­mately 13 years ago, a child from a prominent Eurasian family became ill on a flight from Hong Kong to Manila. She had a hepatoma of the liver which ruptured, causing a nearly fatal hemorrhage. Her family had the plane rerouted to Los Angeles and from there to Pittsburgh, where we performed a liver trans­plantation. Tumor from the lesion was thought to have seeded throughout the abdomen. The prognosis appeared hopeless.

Postoperatively, the child received chemotherapy, and after a stormy course that included a bout of severe pancytopenia, she returned home and grew up. She not only stopped taking immu­nosuppression medication but also became a fashion cover girl and is now on her way to becoming a world-class ballet dancer (as was her mother before her). Like Amie Garrison, this patient has a normal life expectancy. She has acquired tolerance.

Although it was not recognized as such, tolerance has been a theme throughout the entire modern age of organ transplanta­tion. This was belatedly acknowledged at a meeting last March at the University of California, Los Angeles (UCLA). The pur­pose of the conference was to reach a consensus about the mile­stones that had made transplantation a thriving clinical specialty. Eleven early workers in clinical organ and tissue transplantation contributed to the program. Professor Carl Groth (Karolinska In­stitute, Stockholm) was invited to be chairman of the consensus deliberations, the conclusions from which are being published (3).

BUMC PROCEEDINGS 1999;12:253-258

The participants included histocompatibility specialists Jean Dausset, MD, Paul Terasaki, PhD, and Jon J. Van Rood, MD; early investigators of acquired tolerance (Leslie Baruch Brent, PhD) and drug immunosuppression (Robert S. Schwartz, MD); bone marrow transplanters Robert A. Good, MD, and E. Donnall Thomas, MD; and organ transplant surgeons Roy Caine, MD, Joseph E. Murray, MD, Norman E. Shumway, MD, and myself.

The 4 surgeons accounted for the first successful transplan­tations of the kidney, liver, and heart. Murray became a 1990 Nobel laureate. Good performed the first successful bone marrow transplantation in 1968. Thomas was a Nobel laureate in 1990 for his studies of bone marrow transplantation for a variety of diseases. Leslie Brent is the Brent of the famous British team known in the 1950s as the "holy trinity" of transplantation im­munology. Schwartz was the first to show the immunosuppressive qualities of 6-mercaptopurine (6-MP) and azathioprine (4). For his discovery of the first human histocompatibility locus antigen (HLA) (5), Dausset received a Nobel prize. Terasaki and Van Rood introduced tissue matching into clinical transplantation.

MILESTONES VS TURNING POINTS Milestones

The participants placed transplantation milestones in 2 cat­egories (Table 1). The first consisted of generic advances that were applicable to all kinds of allografts. Progressively more ef­fective immunosuppressive agents and drug regimens headed this list. Methods of tissue and organ preservation and histocompat­ibility matching also qualified. Although broadly applicable sur­gical techniques were not listed, they could be exemplified by the development nearly 100 years ago by Alexis Carrel of tech­niques for vascular surgical anastomoses (6).

The second category of milestones was organ- or cell­specific. This consisted of the first examples of long allograft and patient survival (;~6 months) after transplantation of various

From the Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.

Aided by project grant no. OK 29961 from the National Institutes of Health, Bethesda, Maryland.

Presented at grand rounds, Baylor University Medical Center, April 21, 1999.

Corresponding author: Thomas E. Starzl, MD, PhD, University of Pittsburgh Medi­cal Center, 3601 Fifth Avenue, 4C Falk Clinic, Pittsburgh, Pennsylvania 15213.

253

254 BAYLOR UNIVERSITY MEDICAL CENTER PROCEEllINGS VOLUME 12, NUMBER 4

Table 1. Transplantation milestones

I. Generic milestones (applicable to all tissues and organs)

A. First use of individual immunosuppressants (all eventually combined with adrenal cortical steroids) 1. Total body irradiation (1959) 2. 6-MP and azathioprine (1960-1962) 3. Other myelotoxic agents (1960-1965) 4. Adrenal cortical steroids (anecdotally, 1955-1961; systematically,

1962-1963) 5. First effective tolerance-inducing cocktail (azathioprine/prednisone)

(1962-1963)* 6. Antilymphoid antibodies (antilymphocyte globulin) (1966) 7. Cyclosporine (1978-1979) 8. Tacrolimus (1989)

B. Tissue and organ preservation 1. Hypothermia per se

(a) Surface cooling (1960) (b) Cold fluid infusion (1962-1963)

2. Special infusates; slush preservation (a) Lactated Ringer's solution (1962-1963) (b) Low-molecular-weight dextran (1963) (c) Mimicking intracellular composition (1966) (d) Mimicking extracellular composition (1973) (e) UW solution (1987)

3. Artificial postmortem circulation (a) Whole cadaver (1963) (b) Isolated organ (196W

C. Clinical histocompatibility matching 1. Lymphocyte culture LMLR (1963) 2. Serologic (1964) 3. Molecular (1990s)

II. Generic clinical milestones (tissue- or organ-defined milestones: first clinical survival >6 months)

A. Kidney 1. Isografts (1954) 2. Allografts (1959)

B. Bone marrow 1. Isografts (1955) 2. Allografts (1963, 1968)

C. Extrarenal organs 1. Liver (1967) 2. Heart (1968) 3. Lung (1968) 4. Pancreas (1969) 5. Intestine (in multivisceral grafts, 1987; segmental, 1988; complete,

1989)

*Generic strategy applicable with subsequent drugs.

tFirst experiments by A. Carrell and C. A. Lindberg (1935).

kinds of human tisslies and organs. This objective was accom­plished with the kidney in January 1959 (7), and with the liw\" (8), heart (9), lung (10), and pancreas (11) in that order between July 1967 and June 1969. Completely successful bone marrow transplantation was not done until 1968 (12, 13), and intesti­nal transplantation was delayed until the late 1980s.

Turning points The foregoing milestones in both generic and organ-specific

categories, and dozens of lesser ones, afe important. However, as the layers of history were peeled away it became apparent that there actLlally were only 2 seminal turning points in the evolu­tion of clinical transplantation: that allograft tolerance can be acquired and that organs are inherently tolerogenic (14-16). The stage for these turning points had been set during the Second World War with the demonstratiun by Medawar (a basic scien­tist) and Gibson (a plastic surgeon) that rejection is an immune response (17). However, rejection was considered for the ensu­ing decade to be one of the most powerful and irrevocable reac­tions in biology and therefore uncontrollable.

The resulting pessimism about the prospects of clinical trans­plantation had to be modified in 1953 when Billingham, Brent, and Medawar demonstrated the induction of chimerism­associated acquired neonatal tolerance (18). This was the first turning point. The tolerance induced in neonatal mice with sple­nocyte or bone marrow cell infusion mimicked the natural hema­tolymphopoietic chimerism in freemartin cattle reported nearly a decade earlier by Owen (19). The neonatal mouse experiments escalated in a straight line to clinical bone marrow transplanta­tion (12, 13, 20).

The second turning point was the clinical demonstration in the early 1960s that tissue and organ allografts could "self-induce" tolerance when combined with immunosuppression (21). This discovery galvanized a revolution in clinical organ transplanta­tion. The downside, however, was the erroneous conclusion that the "acceptance" and long-term survival of organs occurred by dif­ferent mechanisms than the chimerism-dependent tolerance in­duced by bone marrow and other hematolymphopoietic allografts.

The ostensible differences between hone marrow transplan­tation and organ transplantation (Figure I) seemecl too great to permit any other conclusion. These differences included 1) de­pendence on HLA matching for successful hune marrow trans­plantatiun but not for organ transplantation, 2) risk vs freedom of risk from graft-vs-host disease (C3VHD), 3) frequency vs in­frequency of achievement of drug-free status, and 4) a semantic distinction between the tolerance of bone marrow transplanta­tion and the acceptance of organ grafts.

As it turned out, all of these differences were explained by hematopoietic cytoablation of the bone marrow recipient (ini­tially with total body irradiation, later with cytotoxic drugs) but not of the organ recipient. When this finally was recognized in the 1990s, the linkage between organ transplantation and bone marrow transplantation was established. In addition, the relation of hoth varieties of transplantation to the neonatal tolerance described by Billingham, Brent, and MedawaT was ll[,vious. The epiphany was largely dependent un the demonstration of low­level donor leukocyte chimerism in livLT allograft recipients (14-16). Consequently, it will be useful here to review how liver transplantation evolved.

THE LATE 1950s Turning the clock back more than 40 years, my personal

involvement in transplantation began with the development in 1958 and 1959 of techniques for canine liver transplantation,

OCTOBER 1999 IM~lUNOLOGICAL TOLERANCE: ONE OF BIOLOGY'S MOST INTRIGUING MYSTERIES 255

Solid Organ Bone Marrow

(Medawar)

~-------------------------------------------- ~

Figure 1. The division of transplantation into two separate disciplines, caused by different treatment policies.

alone or as part of multivisceral grafts (22,23), paralleling simi­lar independent investigations in Boston by Francis D. Moore (24). This was at first an exercise in surgical technique. Although adrenal cortical steroids and total body irradiation were known to be immunosuppressive, survival for as long as 3 months had not been achieved in any species with any kind of organ allograft.

Our laboratory efforts took on a different meaning with the demunstration in 1959 by Schwartz and Damashek (4, 25), promptly confirmed by Good's Minnesota team (26), that 6-MP prolonged the survival of skin grafts in rabbits without the need for the kind of bone marrow depression caused by irradiation. When in 1960 CaIne in London (27) and Zukoski in Richmond, Virginia (28), reported that 6-MP therapy prolonged kidney al­lograft survival in dogs, the possibility of similarly treating liver recipients was obvious. In fact, this was attempted clinically less than 3 years later, following advances with immunosuppression made in human kidney recipients.

THE PATHFINDER KIDNEY Between January 26, 1959, and March 1, 1963, the date of

our first attempt to replace a human liver, Murray in Boston (7) and the French teams of Hamburger (29) and Kuss (30) had pro­duced 6 clinical examples of kidney allograft survival exceeding 6 months after pretreatment with total body irradiation (Table 2). Although 4 of these patients soon died, the 2 recipients of fraternal twin kidneys survived more than 20 years. Importantly, the seventh kidney recipient was 11 months posttransplantation by March 1, 1963, after treatment by Murray from the outset (April 1962) with the 6-MP analogue azathioprine (31). This allograft functioned for l7 months.

It had been learned, however, that to achieve more than occasional success in either dogs or humans, azathioprine needed a partner drug. This proved to be dose-maneuverable prednisone (21), which we knew from our canine kidney and liver transplant studies could reverse 90% of the rejections developing under azathioprine. Unbeknownst to us, this steroid effect already had

Table 2. Kidney transplantation::::6 months survival as of March 1963

Case City Date Donor Survival (months)'

Bostont 1-24-59 Frat twin >50

2 Paristt 6-29-59 Frat twin >45

3 Paris 6-22-60 Unrelated§ 18 (died)

4 Paris 12-19-60 Mother§ 12 (died)

5 Paris 3-12-61 Unrelated§ 18 (died)

6 Paris 2-12-62 Cousin§ >13

7 Boston 4-5-62 Unrelated 11

* The kidneys in patients 1,2, and 6 functioned for 20.5, 25, and 15 years, respectively.

Patient 7 rejected his graft after 17 months and died after return to dialysis.

t Boston: J. E. Murray (1 and 7).

It Paris: J. Hamburger (2, 4, and 6); R. Kuss (3 and 5).

§ Adjunct steroid therapy.

Imrnune Reaction

T mURK

Time after Transplantation

~--- ---------------------------------------~

Figure 2. Historical view of events after successful organ transplanta­tion: rejection, its reversal, and the development of donor-specific nonreactivity.

been seen in the autumn of 1960 by Willard Goodwin in a kid­ney recipient at UCLA in whom bone marrow depression had been induced with myelotoxic doses of methotrexate and cyclo­phosphamide (32). Regrettably, the case was not reported until 1963, by which time the pioneer UCLA program had closed down because of the early deaths of all the other recipients.

In the meanwhile, we had begun our clinical kidney trans­plant program in the autumn of 1962, using azathioprine plus prednisone. As reported in October 1963 (21), 8 of our first 10 kidney recipients had prolonged graft survival. This was the first successful series of kidney transplantations. Two of these patients, now old men in their 37th posttransplant year, bear the longest continuously functioning allografts in the world. As has been ex­pected from the observations in dogs, rejection was regularly re­versible. More importantly, there was also clear evidence that the transplanted kidneys had self-induced variable donor-specific tol­erance.

The most convincing evidence of tolerance was the frequent diminution of need for maintenance immunosuppression after the development of rejection and its reversal (Figure 2). The donor-specific nonreactivity was complete enough to allow many patients to go home to an unrestricted environment. The third

256 BAYLOR UNIVERSITY MEDICAL CENTER PROCEEDINGS VOLUME 12, NUMRER 4

100

""'-0 80

C1l > 60 ~ ::::l

(f) 40 C <lJ

~ 20 Il..

0

100

~o 0' 80

C1l 60 > 2: ::::l 40 (f)

'§ 20 (9

0

0

0

• AZA (n~168) .. CYA (n~1835)

• TAC (n~1391)

1 2 3 4 5 Time after Transplantation (years)

2 3

• AZA (n~190) .. CYA (n~2416)

• TAC (n~1582)

4 Time after Transplantation (years)

5

Figure 3. The 3 eras of orthotopic liver transplantation at the Univer­sities of Colorado (1963-1980) and Pittsburgh (1981-1993), defined by azathioprine, cyclosporine, and tacrolimlls (FK 506)-based immune suppression, Similar but less dramatic stepwise improvement has been seen with all organs. Top family of curves: patient survival. Bottom fam­ily: graft survival.

patient in this series stopped all medications about 1 year after transplantation and has heen drug free for more than a third of a century (33).

LIVER TRANSPLANTATION Our early experience with kidney transplantation, combined

with more than 5 years' investigation of canine liver transplan­tation, prompted our attempts to replace the human liver, the first vital extrarenal organ to be transplanted clinically. When the first 3 patients (34), single patients in Boston (35) and Paris (36), and an additional 2 recipients in our Colorado program (37) all died within 23 posttransplant days, clinical liver transplantation ceased worldwide. During the 3 1/2-year self-imposed moratorium, we developed and introduced horse antihuman antilymphocyte globulin clinically, first testing it in kidney recipients as a perioperative adjunct to azathioprine and prednisone (38).

Armed with the encouraging results in the kidney trial, the liver program was restarted in July 1967, using the same 3-drug immunosuppression (azathioprine, prednisone, and antilympho­ctye globulin) that had been tested in kidney recipients. A num­ber oflong survivals were obtained (> 1 year), mostly of children (39). A 33-year-old woman (then a 3-year-old child with biliary atresia and a hel'atuma) is the longest surviving liver recipient in the world, now in her 30th posttransplant year.

Another 13 years would pass before this kind of result be­came regularly attainable. The improvement in transplantation of the liver and other organs has occurred in 3 distinct drug-

Table 3. Central therapeutic dogma

1. Baseline therapy with one or two drugs'

2. Secondary adjustments with steroids or al1tilymphoid agents

3. Case-to-case trial (and potential error) of weaning

*Baseline agents: azathioprine, cyclophosphamide, cyclosporine, cyclosporine­azathioprine, FK 506, FK S06-azathioprine.

defined eras: azathioprine-based therapy, cyclosporine-based therapy (40, 41), and tacrolimus (FK 506) (42) (Figure 3). Be­cause retransplantation became increasingly more reliable with the better immunosuppression, patient survival was successively better than graft survival. With the advent of tacrolimus, the first immunosuppressant to be evaluated primarily with liver trans­plantation, intestinal transplantation hecame a viable clinical option.

EXPLANATION OF "ORGAN ACCEPTANCE" Despite the diversity of azathioprine, cyclosporine, tacro­

linms, and other immunosuppressants, the basic pattern of im­munologic confrontation and involution remained the same. With individualized treatment adjustments guided by evidence of graft rejection, graft acceptance could be engineered as de­scribed in Table 3. What was being accomplished with this strat­egy? The answer was discovered in 1992.

In the spring of 1992, 25 liver and 5 kidney recipients who had survived with functioning grafts for 10 to 30 years were brought to Pittsburgh for restudy. In addition to blood sampling, open biopsy was obtained of the transplanted organs; of the re­cipient lymph nodes and skin; and, when indicated, of other host organs, including the heart, intestine, and bone marrow. Small numbers of donor leukocytes were found in the host peripheral lymphoid and nonlymphoid tissues or blood of all 30 patients (14, 15). The donor cells were identified by study of the HLA anti­gens or sex karyotype with immunocytochemical methods. The findings were confirmed with polymerase chain reaction studies. The chimerism included the prominent presence of dendritic cells.

Now, we realized that organ transplantation involved a double immune reaction-which had both a host-vs-graft re­sponse and a covert graft-vs-host response (Figure 4). Because the dominant imrntme reaction following organ transplantation usu­ally is that of the host, the common complication is rejection of the graft. However, serious or lethal GVHD is not rare after trans­plantation of leukocyte-rich organs like the liver. For allograft acceptance (and tolerance) to occur (rather than rejection or GVHD), the seminal tolerogenic mechanism was postulated to be "[widespread] responses of coexisting donor and recipient im­mllne cells, each to the other, causing reciprocal clonal expan­sion, followed by peripheral clonal deletion" (14, 15).

By 1998, compelling evidence had accumulated from con­trolled animal experiments confirming this hypothesis, as sum­marized in a recent review coauthored with Rolf Zinkernagel (16). Organ and bone marrow "acceptance" were related forms of acquired tolerance-not fundamentally different than the

OCTOBER 1999 IMMUNOLOGICAL TOLERANCE: ONE OF BIOLOGY'S MOST INTRIGUING MYSTERIES 257

Failure

Immune ;;~~~~;;~~~~ Reaction I-S:~-------=,=== Success

----)0. Failure

Time after Transplantation

Figure 4. Contemporaneous host-vs-graft and graft-vs-host reactions that occur after transplantation. Treatment failure is defined as the inability to cuntrol one of the reactions or sometimes both. Acute re­ciprocal clonal exhaustion after successful transplantation is maintained subsequently by chimerism-dependent low-grade stimulation of hoth leukocyte populations that may wax and wane.

Q) (/) c: 8. (/) Q)

a: OJ co ::J

E E

(1) Maintenance (3) ...... ,..... clonal

",deletion ~~ -', ',-------------...---,-

............... ·····················(·;··t·; .. ········· ........................... .

Leucocyte Depletion (4)

Figure 5. The 4 events that occur in close temporal approximation when there is successful organ engraftment: ahovt:', double acute clonal exhaustion (numbers 1 and 2) and subsequent maintenance clonal exhaustion (3) plus, below, loss of organ immunogenicity due to deple­tion of the graft's passenger leukocytes (4).

major histocompatibility (MHC)-restricted antigen-specific tol­erance that can be induced by non cytopathic viruses and other microorganisms (16). However, the response to an organ allograft is made more complex than that to a noncytopathic microorgan­ism by the presence of immunocompetent donor cells and the consequent double immune reaction (graft vs host as well as host vs graft).

Clonal exhaustion and an ancillary mechanism of "immune indifference," both regulated by the migration and localization of the donor leukocytes, were responsible for the 4 interrelated events shown in Figure 5. These must occur in close temporal proximity for successful organ engraftment: double acute clonal exhaustion; maintenance clonal exhaustion, which waxes and wanes; and loss of organ immunogenicity as the passenger leu­kocytes depart from the graft.

The reciprocal nullification of the interreactive immune re­sponses explained the poor prognostic value of tissue matching for organ transplantation (43). This nullification effect also ex-

Table 4. Effectors involved in response to cytopathic parasites and discordant xenografts

The first line of defense

Interferons

M acrophages

y /8 T cells

Natural killer (NK) cells

B cells

Nonspecific or less specific effectors

Complement Early interleukins

Phagocytes

iii

plains why GVHD is so uncommon, even after organ transplan­tation with engraftment of leukocyte-rich organs like the liver and intestine, and why it is safe to infuse adjunct donor bone marrow in organ recipients providing the patients are not immunologically weakened in advance by cytoablation or other means (15, 33).

It follows that conventional bone marrow transplantation is a mirror image of the events after organ transplantation and is also governed by antigen migration and localization. The host leukocytes are not all eliminated by pretransplant cytoablation (44, 45). The weak host-vs-graft reaction mounted by those re­maining recipient cells and the parallel graft-vs-host reaction of the dominant population of donor cells can eventually result in reciprocal tolerance (16,46). It might be added that these same events transpire in the historically important "mixed chimerism" tolerance models. We had in fact returned full circle to the first observations of natural tolerance in freemartin cattle reported in Science 55 years earlier by Ray Owen ( 19).

XENOTRANSPLANTATION There is no MHC-restricted safety valve for cytopathic mi­

croorganisms, which arc typically extracellular and generate the full resources of the innate as well as the adaptive immune sys­tem (16, 47, 48). An uncontrollable innate immune response involving the effectors shown in Table 4 is provoked by discor­dant xenografts expressing the Cal-a Cal epitope, an epitope that is also found on numerous cytopathic bacteria, protozoa, and viruses. The clinical use of such discordant animal donors (e.g., pigs) will require either changing the xenogeneic epitope to one that mimics a noncytopathic profile or eliminating the xenoge­neic epitope (16,49).

SUMMARY Thus, the 2 seminal turning points that allowed the clini­

cal emergence of transplantation turned out to invoke the same, not different, tolerance mechanisms. This has explained histori­cal enigmas, including the meaning of allograft acceptance, which is simply a form of acquired tolerance. This paradigm also establishes a better context for the design of experiments and therapies yet to come.

References 1. Ramos He, Reyes j, Abu-Elmagd K, Zeevi A, Reinsmoen N, Tzakis A,

Demetris AJ, Fung JJ, Flynn B, McMichael J, Ebert F, Starzl TE. Weaning of immunosuppression in long-term liver transplant recipients. Transplan­tation 1995;59:212-21 7.

258 BAYLOR UNIVERSITY MEDICAL CEl\TER PROCEEDINGS VOLUME 12, NUMBER 4

2. Mazariegos GV, Reyes J, Marino IR, Demetris AJ, Flynn B, Irish W, McMichael J, Fung JJ, Star:! TE. Weaning of immunosuppres>ion in liver ttalbl'lant recipients. Transplantation 1997;63:243-249.

3. Groth CO, Brent LB, CaIne RC, DaussetJ, Good RA, Murray JE, Shumway NE, Schwartz RS, Stad TE, Terasaki PI, Thomas ED, Van Rood JJ. His­tori cal landmarks in clinical transplantation: conclusions from the consen­sus c.lIlference held at University of California, Los Angeles (UCLA). World] Surg. In press.

4. Schwartz R, Dameshek W. Drug-induced immunological tolerance. Nature 19'i<J;tn: 1682-1683.

5. Dausset]. Iso-leuco-anticorps. Acta HaematoI1958;20:156-166. 6. Carrel A. The operative technique Il)r vascular anastomoses and transplan­

tation of viscern. bon Medicine 1902;98:R59-864. 7. Merrill JP, Muml)' JE, Harrison JH, Friedman EA, Dealy JB Jr, D:l III min GJ.

Successful homotransplantation of the kidney between non-identical twins. N Engl} Med 1960;262:1251-1260.

8. Star:l TE, Gwth CG, Brcmchneider L, Moon JB, FlIlginiti VA, Cotton EK, Porter KA. Exrended survival in.3 cases of orthotopic homotransplan' tation of the human liver. Surgery 1968;63:549-563.

9. Barnard CN. What we have learned about heart transplants. ] Thorac Cardiol'asc Surg 1968;56:4')7-468.

10. Derom F, Barbier F, RingOlr S, Versieck J, Rolly U, Berzsenyi U, Vermeire P, Vrints L. Ten-month survival after lung homotransplantation in man.] Thorac Cardiovasc Surg 1971 ;61 :835-846.

11. Lillehei RC, Simmons RL, Najarian JS, Wei! R, Uchida II, Ruiz JG. Kjcllstrand CM, GLletz Fe. Pancreatico·duodenal allotransplantation: ex­perimental and clinical experience. Ann Surg 1970;172:405-436.

12. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution ,)f sex· linked lymphopenic immun<liogical deficiency. Lan· cet 1968;2:1366-1.369.

13. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet 1968;2: 1364-1366.

14. Statzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism, and graft acceptance. Lancet 1992;339:1579-1582.

15. Stad TE, Demctris AJ, Trucco M, MurClse N, Ricordi C, Ildstad S, Ramos H, Tollo S, Teakis A, Fung JJ, Nalesnik M, Zeevi A, RlIdert WA, Kocov" M. Cell migration and chimerism after whole-organ transplantation: the hasis of graft acceptance. Hepatulogy 1993;17:1127-1152.

16. Star:l TE, Zinkcrnagel RM. Antigen localization and migration in immu­nity and tolerance. N Eng!] Med 1998;339: 1905-1913.

17. Gibson T, Medawar PB. The fate of skin homografts in man. ] Anatomy 1943;77:299-310.

18. Billingham RE, Brent L, lvledawar PB. "Actively acquired tolerance" offm· eign cells. Nature 1953; 17 2:603-606.

19. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945;102:400-401.

20. Thomas ED, Stmh R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H, Buckner CD. Bone-marrow transplantation. N Engl] Med 1975;292:832-843, 895-902.

21. Scad TE, Marchioro TL, Waddell WR. The reversal of rejection in lou· m:m renal homografts with subsequent development of homograft toler· ance. Surg Gynecol Ohstet 1963;117:385-395.

22. Starzl TE, Kaupp HA Jr, Brock DR, Lazarus RE, Johnson RV. Reconstruc­tiYe problems in canine liver homotransplantation with special reference to the postopcrJtive role of hepatic "cnous flow. Surg GYllecol ObstcL 1960;111:733-743.

2.3. Stm21 TE, Kaupp HA Jr. Mass homotransplantation of abdominal organs in dug,. Surg F01um 1960; 11 :28-30.

24. Moore FD, Wheeler HB, Dimissianos HV, Smith LL, Balankura G, Abel K, Greenberg JB, Dammin GJ. Experimental whole organ transplantation of till: liver and of the spleen. Ann Surg 1960; 152: 374-387.

25. Schwartz R, Dameshek W. The effects uf 6-merGlptopurine on homograft reactions. ] CUn Invest 1960;39:952-958.

26. Meeker W, Condie R, Weiner 0, Varco RL, Oood RA. Prolongation of skin homograft sllnival in rabhits hy 6.mcrcaptopurine. Pmc Soc Exl) fliol Med 1959; 102:459-461.

27. Caine RY. The rejection of renal homografts: inhibition in dogs by 6-mer­C:lptopurine. Lancet 1960; 1 :417-418.

28. Zukoski CF, Lee HM, Hume [)M. The prolongation ,If functiunal survi\':,] of canine renal homografts by 6-mercaptopurine. Surg Forum 1960; 11:470-472.

29. Hamhllfger J, Vaysse J, Crosnier J, Auvert J, Labnne CL, Hopper J Jr. Re:· nalhomotransplantation in man after rCidiation 01' the recipient. Am] Med 1962;32:854-871.

30. Kuss R, Legrain M, Mathe G, Nedey R, Camey M. Homologous human kidney transplantation. E:-.:perience with six patients. Postgrad Med] 1962; 38:528-53L

31. Murray JE, Merrill JP, Harrison JH, Wilson RE, Dammin GJ. Prolonged sur­vi\'al of human· kidney homografts by immunosuppressive drug therapy. N Engl} Med 1963;268:1315-1323.

32. Goodwin WE, Kaufman]J, Mims MM, Turner RD, Glassock R, Goldman R, Maxwell MM. HlIlnan renal transplantation. l. Clinical experience with six cases of renal homotr:msplantatiun . .T Urolog~ 1963;89: 13-24.

33. Starzl TE, Demetris AJ, Murase N, Tflicco M, Thomson A''\i/, Ran AS. The lost chord: microchimerism and allograft survival. Immunol Today 1996; 17:577-584, discussion 588.

34. Star:! TE, Marchioro TL, Von Kaulla KN, Hermann G, Brittain RS, W"ddell WR. Homotransplantation of the liver in humans. ~!<Tg Gynecol Obstet 1963;117:659-676.

35. Moore FD, Birtch AG, Dagher F, Veith F, Krisher JA, Order SE, Shllcart WA, Dammin GJ, Couch NP. Immunosuppression and vascular insuffi· ciency in liver transplantation. Ann NY Acad Science 1964;120: 729-738.

36. Demirleau, Noureddine, Vignes, Prawerman, Reziciner, Larraud, Louviers. Attempted hepatic homogmft [article in French]. Mem Acad Chir (Pari,) 1964;90:177-179.

37. Statzl TE, Marchioro TL, Rowlands DT Jr, Kirkpatrick CH, Wilson WEe, Ritkind 0, Waddell WR. Immunosuppression after experimental and clini­cal hClmotransplantatioll of the liver. Ann Surg 1964; 160:411-439.

38. Stad TE, Marchioro TL, Porter KA, Iwasaki Y, Cerilli OJ. The lise ofhet· erologou, antilymphoid agents in canine renal and liver homotransplan­tation and in human renal homotransplantation. Surg Gynecol Obstet 1967; 124:301-308.

39. Starzl TE, Groth CO, Brettschneider t, Penn I, Fulginiti VA, Moon JB, Blanchard H, Martin AJ Jr, Porter KA. Orthotopic homotransplantation of the human liver. Ann Surg 1968; 168:392-415.

40. CaIne RY, White OJ, Thiru S, Evans DB, McMaster P, Dunn DC, Cradduck UN, Pentlow BO, Rolles K. Cyclosporin A in patients receiving renal al­lografts from cadaver donors. Lancet 1978;2: 1323-1327.

41. Stmzl TE, Klintmalm GB, Porter KA, hvatsllki S, Schwter GP. Liver trans­plantation with lise of cyclosporin A and preclnisone. N Engl] Med 1981;305:266-269.

42. Statzl TE, Todo S, Fung J, Demetris AJ, Venkataramman R, Jain A. FK 506 for liver, kidney, ,md pancreas transplantation. Lallcet 1989;2: 1000-1004.

43. Starzl TE, Ral) AS, Twcco M, Fontes P, Fung JJ, Demetri, AJ. Explana­tion for loss of the HLA matching effect. Transplant Proc 1995;27:57-60.

44. Przepiorka 0, Thomas ED, Durnam DM, Fisher L. Use of a probe to repeat sequence of the Y chromosome for detection of host cells in peripher,'] hlood of bone marrow transpl:ll1t recipients. Am] CUn PathoI1991;95:201-206.

45. Wessman M, Popp S, Ruutu T, Volin L, Cremer T, Knuutila S. Detection of residual host cells after bone marrow transplantation using non· isotopic in situ hybridization and karyotype analysis. Bone Marrow Tramplant 1993; 11:279-284.

46. Stad TE, Demetris AJ. Transplantation milestones. Viewed with one· and two·way paradigms of toler'lllce. ]AMA 1995;27.3:876-879.

47. Zinkernagel RM.lmmunology taught by viruses. Science 1996;271: 173-178. 48. Zinkernagel RM, Ehl S, Aichele P, Oehen S, Kundig T, Heng<lrtner H.

Antigen 10caiisCition regubtes immune responses in a d,JSc, and time· dependent fashion: a geographical view of immune reactivity. Immunol Re,' 1997; 156: 199-209.

49. Starzl TE, Rao AS, Murase N, FungJ, Demetris AJ. Will xenotransplantation en'r be feasible!.1 Am Coll Surg 1998; W6:383-387.